SUCLG2 Regulates Mitochondrial Dysfunction through Succinylation in Lung Adenocarcinoma

Abstract Mitochondrial dysfunction and abnormal energy metabolism are major features of cancer. However, the mechanisms underlying mitochondrial dysfunction during cancer progression are far from being clarified. Here, it is demonstrated that the expression level of succinyl‐coenzyme A (CoA) synthetase GDP‐forming subunit β (SUCLG2) can affect the overall succinylation of lung adenocarcinoma (LUAD) cells. Succinylome analysis shows that the deletion of SUCLG2 can upregulate the succinylation level of mitochondrial proteins and inhibits the function of key metabolic enzymes by reducing either enzymatic activity or protein stability, thus dampening mitochondrial function in LUAD cells. Interestingly, SUCLG2 itself is also succinylated on Lys93, and this succinylation enhances its protein stability, leading to the upregulation of SUCLG2 and promoting the proliferation and tumorigenesis of LUAD cells. Sirtuin 5 (SIRT5) desuccinylates SUCLG2 on Lys93, followed by tripartite motif‐containing protein 21 (TRIM21)‐mediated ubiquitination through K63‐linkage and degradation in the lysosome. The findings reveal a new role for SUCLG2 in mitochondrial dysfunction and clarify the mechanism of the succinylation‐mediated protein homeostasis of SUCLG2 in LUAD, thus providing a theoretical basis for developing anti‐cancer drugs targeting SUCLG2.

H292 (C), H23 (D), HCC827 (E), and SPC-A1 (F) and the human lung epithelial cell line BEAS-2B (G) were transfected with either individual siRNAs targeting SUCLG2 or control siRNAs.Twenty-four hours later, cells were seeded in 24-well plates.At       siRNAs, and co-IP and western blotting were used to detect the ubiquitination level of SUCLG2.(J and K) SIRT5 was overexpressed (J) or knocked down (K) in A549 cells transfected with the His-SUCLG2 K93R plasmid, and the cells were treated with CHX (20 μg/mL) for 0h, 3h, 6h, 9h, 12h, and 24 h.The degradation rate of the SUCLG2 protein was detected by western blotting.
, cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet.The dye was extracted with 10% acetic acid, and the relative proliferation was assessed based on the increase in absorbance at 595 nm (upper panels).Western blotting was used to determine the knockdown efficiency (bottom panels).The data represent the average of three independent experiments (mean ± SD). ***P < 0.001, **P < 0.01, ns: P > 0.05.(H-J) LUAD cell lines A549 (H), SPC-A1 (I), and H1299 (J) were transfected with either individual siRNAs targeting SUCLG2 or control siRNAs.A colony formation assay (left-hand panels) was performed and quantitatively analyzed using ImageJ software.The results represent the average of three independent experiments (mean ± SD). **P < 0.01, ***P < 0.001 (right-hand panels).

Figure
Figure S2 SUCLG2 knockout affecting the metabolism of LUAD cells.(A and B) A549-SUCLG2-KO and A549-WT cells were used for untargeted positive ion mode

Figure S3 .
Figure S3.Succinylation affects the degradation of ACOT9 and ME2.(A and B) The indicated plasmids were transfected into A549 cells.Co-IP and western blot were used to detect the succinylation level of ACOT9 (A) and ME2 (B).(C and D) A549 cells were transfected with the indicated plasmids, then treated with CHX (20 μg/mL) for 0h, 3h, 6h, 9h, 12h, and 24 h.The degradation rate of ACOT9 (C) and ME2 (D) western blotting.(E-G) A549-WT and A549-SUCLG2-KO cells were transfected with the indicated plasmids, then treated with CHX (20 μg/mL) for 0h, 6h, 12h and 24 h.The degradation rate of ACOT9 and ME2 mutant proteins were detected by western blotting.

Figure S4 .Figure
Figure S4.The mRNA level of SUCLG2 is not significantly upregulated in LUAD.(A) The mRNA level of SUCLG2 was detected in LUAD cell lines and the normal epithelial cell line BEAS-2B with RT-PCR.The data represent the average of three independent experiments (mean ± SD). ns, P > 0.05; ***P < 0.001.(B) SUCLG2 TPM expression in LUAD tissues and adjacent tissues was analyzed by GEPIA (http://gepia.cancer-pku.cn).

Figure S6 .
Figure S6.p62 plays an important role in the protein degradation of SUCLG2.(A) The co-localization of SUCLG2 and p62 in A549 cells was detected by immunofluorescence.(B and C) A549 cells were transfected with the pCMV-Flag-p62 (B) or pcDNA-His-SUCLG2 plasmid (C), and the interaction between SUCLG2 and p62 was detected using co-IP and western blotting.(D and E) Flag-p62 was transfected into A549 (D) and H1299 (E) cells for 48 h, and the protein level of SUCLG2 was detected by western blotting.(F) A549 cells were transfected with the indicated plasmids, followed by co-IP and western blotting.(G) Co-IP was performed in A549 cells transfected with SUCLG2 and TRIM21 before subjecting them to western blotting with an anti-p62 antibody.

Figure
Figure S7.SIRT5 is the desuccinylase for SUCLG2.(A) Flag-SIRT7 and His-SUCLG2 were transfected into A549 cells, and the succinylation of SUCLG2 was detected by western blotting.(B and C) Flag-SIRT5 and His-SUCLG2 were